Pamela K. Geyer - US grants

Insulators possess two basic properties; l) they disrupt enhancer-promoter interactions only when inserted between these control elements and 2) they prevent chromosomal position effects. As such, insulators impart limitations to regulatory elements within chromosomes. Two general models have been proposed for insulator function; those in which insulators organize changes in higher-order chromatin structure to block regulatory interactions (domain boundary model) and those in which insulator proteins engage in local interactions with transcriptional proteins to intercept control signals (decoy model). To elucidate the molecular mechanisms involved in insulator action, we will study the gypsy insulator, which was isolated from the gypsy retrotransposon. This insulator provides an excellent model, as it is the only known insulator whose function requires a single DNA binding protein, the Suppressor of Hairy-wing [Su(Hw)] protein, to bring insulator complexes to the chromosome and whose effects can be manipulated genetically. In addition to binding the gypsy insulator, the Su(Hw) protein associates with nearly 200 non-gypsy sites within euchromatin. We will distinguish between the two prevailing models of insulator function by defining the mechanisms involved in blocking enhancer-promoter communication and prevention of heterochromatic repression. These studies will determine the effects of the gypsy insulator on the expression of defined transgenes into which the insulator is placed. This approach was chosen over those that study the bulk properties of the Su(Hw) protein because the nature of the non-gypsy target sites is unknown. To understand the function of the Su(Hw) protein at these sites, the non-gypsy target sites will be isolated and we will determine whether they possess insulator function. Our data will elucidate mechanisms used to define regulatory interactions within the genome, thereby providing insights into fundamental questions of how enhancer-promoter interact and the effects of chromatin structure on gene expression. Finally, our studies will enhance the development of better strategies to deliver dependable gene expression in gene therapies, as insulators overcome a major obstacle confronting such technologies, chromosomal position effects.

DESCRIPTION (provided by applicant): Insulators are conserved genomic elements that organize chromosomes into independent functional domains to maintain transcriptional fidelity. These elements protect gene expression from positive and negative regulatory effects by blocking interactions between enhancers, silencers, and promoters. The goal of our studies is to understand molecular mechanisms used by insulators to organize eukaryotic genomes into transcriptional domains. In studies described herein, the Drosophila gypsy insulator will be used as model to elucidate mechanisms of insulator action. The gypsy insulator is a well-studied insulator that was isolated from the gypsy retrovirus. This insulator is the only one known that requires a single DNA binding protein, the Suppressor of Hairy-wing [Su(Hw)]. Once bound to chromosomes, the Su(Hw) protein recruits the BTB/POZ domain protein, Modifier67.2, to generate a protein complex that establishes insulator activity. Mutations in the genes encoding the gypsy insulator proteins are available, making the system amenable to genetic manipulation. The gypsy insulator proteins associate with hundreds of genomic sites that are not sites of gypsy retroviral insertion. These observation suggest that Su(Hw) and Modifier67.2 have global roles in establishing independent regulatory domains in the Drosophila genome. We will test this hypothesis by determining the DNA and proteins requirements of endogenous Su(Hw) sites and define their role in organization of regulatory domains throughout chromosomes. These data will provide insights into mechanisms used to delimit transcriptional regulatory domains within eukaryotic genomes, thereby providing insights into fundamental questions of control of enhancer and silencer function. Understanding mechanisms of insulators action has therapeutic utility for gene therapy, as inclusion of insulators within gene transfer vectors can improve the expression of transgenes, while preventing inappropriate regulatory effects of gene transfer vectors on endogenous gene expression.

Telomeres are composed of two discrete regions, a terminal array of a simple G-rich repeat and an adjacent subtelomeric region containing more complex arrangements of middle and highly repetitive DNA. The terminal G-rich sequences are required for chromosomal integrity and stability. The function of the subtelomere in cellular processes is unknown. Studies of the human disease facioscapulohumeral muscular dystrophy (FSHD) may provide insights into this question. FSHD is an autosomal dominant neuromuscular disease caused by deletion of integral copies of D4Z4, a subtelomeric tandem repeat organized in a large array on 4q. The critical FSHD gene is unknown. Analysis of D4Z4 suggests that it participates in the formation of telomeric repressive heterochromatin. We propose that shortening of the D4Z4 array impairs formation of a domain of repression and results in activation of neighboring gene expression. We will use two approaches to explore this proposed role for D4Z4 in transcriptional regulation. First, we will determine whether expression of adjacent genes increases upon removal of D4Z4 through a comparison of mRNAs from FSHD and control muscle. Second, we will directly test for D4Z4 mediated repression by determining whether D4Z4 confers transcriptional repression to a reporter gene in a model organism. Results from these experiments will provide an understanding of the role of the subtelomere on adjacent gene expression. As the human genome contains a high concentration of genes in telomeric regions, subtelomeric control of transcription may be important in the pathogenesis of other disease phenotypes.

[unreadable] DESCRIPTION (provided by applicant): The University of Iowa Medical Scientist Training Program (MSTP) is a structured, yet flexible Program, with a mission to provide outstanding training in both clinical medicine and scientific investigation for well-qualified students who are interested in pursuing careers as physician-scientists. In its 27-year history, 109 graduates have received MD/PhD degrees. Of these graduates, 77 have completed postgraduate training, with the majority of these individuals engaged in scientific investigation at major academic medical centers or pharmaceutical companies. Our graduates are making important contributions, both by training future physician-scientists and by increasing the understanding of human disease. [unreadable] [unreadable] Iowa MSTP students are selected from a national pool of highly qualified applicants, who have a demonstrated record of scientific involvement. The Iowa MSTP is an integrated training program, such that students are never just medical students or just graduate students. This is accomplished by several thoughtfully designed initiatives that connect scientific investigation with clinical medicine. These activities both enhance the education of our trainees and invigorate the entire scientific community. The MSTP capitalizes on a dynamic training faculty and benefits from the support of the Dean of the Carver College of Medicine, who recognizes the unique role of physician-scientists in academic medical centers. [unreadable] [unreadable] Currently, the Iowa MSTP has 56 trainees (36% women and 7% URM). These students are in various stages of training, including 20 students in the pre-clinical medical school curriculum, 31 students in the graduate phase of training, and 5 students in the clinical phase of training. MSTP students are leaders among their peers in medical and graduate school. [unreadable] [unreadable] Continued support of the Iowa MSTP is requested for years 29 to 33 (2005 to 2010). We request support for 22 trainees in year 29, 24 trainees in year 30, and 26 trainees in each of years 31, 32, and 33. This request reflects an increase from our current level of support of 17 trainees per year. Our request is based on our strong applicant pool, the high quality of our matriculants, the reputation of our outstanding training faculty and the success of our graduates. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Abstract: The nuclear lamina is a protein meshwork underneath the nuclear envelope (NE) that contributes to chromosome organization and gene regulation. One class of lamina proteins shares an ~40 amino acid LEM domain (LEM-D) that binds Barrier-to-Autointegration Factor (BAF), the conserved chromatin bridging protein. Mutations in genes encoding LEM-D proteins cause several human diseases known as laminopathies, including Emery-Dreifuss muscular dystrophy, cardio- myopathies and bone density disorders. These pathologies are tissue-restricted, even though the relevant LEM-D proteins are broadly expressed. Emerging data suggest that laminopathies arise from defects in homeostasis of mesenchymal stem cell populations. Our studies will define the function of LEM-D proteins in Drosophila to elucidate the role of the nuclear lamina in conferring tissue-specific regulation during development. These studies capitalize on our genetic isolation of mutations in three genes encoding LEM-D proteins. Our investigations have shown that the Drosophila LEM-D proteins have unique and overlapping developmental requirements, with evidence of age-enhanced phenotypes and a role in the morphogenesis of a mesenchymal stem cell niche. In this proposal, three aims are proposed. First, we will define requirements for dBAF during development, to understand how this chromatin binding protein contributes to the interphase functions of LEM-D proteins. Second, we will determine how the LEM-D is used to establish tissue- specific functions of this class of lamina proteins. Third, we will establish how LEM-D proteins contribute to critical regulatory pathways involved in the morphogenesis of the germline stem cell niche. Together, these investigations elucidate how BAF and LEM-D proteins work together in the NE to establish distinct nuclear lamina functions required for tissue development. As such, these studies will provide insights into molecular mechanisms of human laminopathies.